Corn is used in a broad range of applications; from ethanol and animal feed production to production of products directly for human consumption, for example breakfast cereal. On average, 20% of corn produced in the U.S. is used for domestic food and industrial purposes. Material output from processed corn includes starch for direct use or chemical modification, starch used as a degradative feedstock for the manufacture of ancillary products, and coproducts/byproducts such as gluten feed, gluten meal and corn oil. As the list of products containing corn-derived ingredients grows, so does the percentage of the corn that is utilized by the corn processing industry.
A central component in the direct, or indirect, use of corn for many products is starch. The central importance of starch to plant development and to food, feed, and industrial markets has motivated researchers across many years to look for mechanisms that control starch biosynthesis. Mutants of maize that affect seed starch deposition have been instrumental in characterizing the biochemistry of starch synthesis. Considerable research effort continues to explore the metabolic systems involved in synthesizing starch, but in addition molecular techniques are being used to analyze and study genes that encode enzymes known to be critical in starch biosynthesis. In discovering which regions of the genes encode metabolism-controlling aspects of the enzymes, scientists are beginning to manipulate starch metabolism through genetic engineering.
The use of seed starch mutants in various crop plants and the production of transgenic plants that over- or under-express specific proteins has indicated that many proteins/enzymes are capable of affecting starch biosynthesis in storage organs. This can occur directly, by impacting the proteins that: (1) produce the substrate(s) for starch synthesis, (2) initiate the glucose polymerization process and elongate the structure into macromolecules, or (3) alter the structure of the polymers once the elongation process has begun. In addition to a direct impact on starch metabolism, starch production can also be negatively impacted by dysfunction or deficiency of proteins that are catalysts in sugar metabolism or act as transporters of intermediary compounds. Proteins involved in assimilate transport, such as the brittle-1 protein of maize endosperm amyloplast membranes, sucrose carrier proteins or others homologous to the hexose transporter of the chloroplast can also affect starch synthesis by restricting the availability of substrates for normal starch and/or sugar metabolism.
Sucrose is considered to be the primary metabolite utilized in the synthesis of starch, although seed grown in vitro with the reducing sugars, glucose or fructose, also produce starch. In simple terms, the sugars are converted into the sugar nucleotides, ADP-glucose and UDP-glucose, either directly or via phosphorylated carbohydrate intermediates. The sugar nucleotides are substrates for the synthase enzymes that polymerize the glucosyl portion of the molecules into long chains of glucose. The polymers remain essentially linear (amylose) or become branched (amylopectin) and combine in a specific fashion to become granules of starch. The proportion of sugar and other carbohydrates, protein, and oils in seeds and fruits is controlled, at least in part, by the conversion of hexose sugars to hexose-6-phosphate by hexose kinase. The hexose kinases can be divided into three general categories according to their hexose substrate specificity. Hexokinase (HK) can phosphorylate glucose and fructose, while glucokinase (GK) and fructokinase (FK) are relatively specific for the respective hexose isomer. Most genomes contain multiple hexose kinases and multiple isozymes of the enzymes. Many plant tissues express hexose kinases.
The hexose sugars used in the production of starch in the seed are moved in the phloem primarily as sucrose. Sucrose is the primary energy source moved from photosynthetic portions of the plant to areas where the energy is utilized or storage of the energy occurs. Sucrose is translocated from the phloem to the seed via specialized cells. Once to the seed, the sucrose (a disaccharide) is often broken down into monosaccharides. Hexokinases add a high energy phosphate to the monosaccharides making hexose sugars available for use in catabolic and anabolic pathways. It has recently been shown that hexokinase expression in an entire plant (driven by the constitutive cauliflower mosaic virus 35S promoter, CaMV35S) leads to growth repression and decreased true leaf development (Xiao, et al., Plant Molecular Biology 44:451, 2000). It has also been shown that overexpression of hexokinase driven by the CaMV 35S promoter inhibits growth, reduces photosynthesis, and induces rapid senescence in tomatoes (Dai, et al., Plant Cell 11:1253, 1999). When these published data are taken together they show that expression of hexokinases under a constitutive promoter is not advantageous for obtaining plants with increased yield and suggest that an overexpression of hexokinase in seed would lead to seed senescence. The present invention, however, shows that the targeted expression of a fungal hexokinase to seeds does not lead to seed senescence, but rather leads to the augmentation of specific aspects of yield, for example, measures of starch per seed.